Gravity-Driven Microfluidic Siphons: Fluidic Characterization and Application to Quantitative Immunoassays

A range of biosensing techniques including immunoassays are routinely used for quantitation of analytes in biological samples and available in a range of formats, from centralized lab testing (e.g., microplate enzyme-linked immunosorbent assay (ELISA)) to automated point-of-care (POC) and lateral flow immunochromatographic tests. High analytical performance is intrinsically linked to the use of a sequence of reagent and washing steps, yet this is extremely challenging to deliver at the POC without a high level of fluidic control involving, e.g., automation, fluidic pumping, or manual fluid handling/pipetting. Here we introduce a microfluidic siphon concept that conceptualizes a multistep ″dipstick″ for quantitative, enzymatically amplified immunoassays using a strip of microporous or microbored material. We demonstrated that gravity-driven siphon flow can be realized in single-bore glass capillaries, a multibored microcapillary film, and a glass fiber porous membrane. In contrast to other POC devices proposed to date, the operation of the siphon is only dependent on the hydrostatic liquid pressure (gravity) and not capillary forces, and the unique stepwise approach to the delivery of the sample and immunoassay reagents results in zero dead volume in the device, no reagent overlap or carryover, and full start/stop fluid control. We demonstrated applications of a 10-bore microfluidic siphon as a portable ELISA system without compromised quantitative capabilities in two global diagnostic applications: (1) a four-plex sandwich ELISA for rapid smartphone dengue serotype identification by serotype-specific dengue virus NS1 antigen detection, relevant for acute dengue fever diagnosis, and (2) quantitation of anti-SARS-CoV-2 IgG and IgM titers in spiked serum samples. Diagnostic siphons provide the opportunity for high-performance immunoassay testing outside sophisticated laboratories, meeting the rapidly changing global clinical and public health needs.


Fluorescence Alkaline Phosphatase (AP) amplification with substrate, AttoPhos
High fluorescence product Low fluorescence substrate   (Table S2 and Figure S2B) was computed from the ratio d c = 4A/P, with the cross sectional area A and perimeter P measured in ImageJ (NIH, USA) from microphotographs of cross sections of the MCF taken with a AMG EVOS microscope (catalogue no: AMEFC4300, Thermo Fisher Scientific, Massachusetts, USA).
Then temperature was increased to 95°C for 30 minutes until all the PVOH had dissolved. As some evaporation occurred during the dissolution process, the volume of solution was adjusted by adding extra ultra-Pure water as required and 0.05% (w/w) Proclin 300 (catalogue no: 48912-U, sourced S4 from Sigma-Aldrich, Dorset, UK) added to prevent microbial growth. The hydrogel coating started by injecting 1.5 meter long MCF strips with the PVOH solution using a push-fit, PEEK luer connector developed in house [1] with a 25mL plastic syringe and left to incubate for 2 hours, after which the solution was blown out using a 25mL syringe filled with air. This was followed by gentle washings with Ultra-Pure water and 0.05% (w/w) Tween 20 (catalogue no: P1379, sourced from Sigma-Aldrich, Dorset, UK) at room temperature (20 o C) with a 25mL syringe, ensuring all capillaries were filled with liquid. Finally, all liquid was again withdrew from the microcapillaries and the MCF strip trimmed to produce shorter, 150mm long strips (or as required) that were dried again individually before storage in sample bag. The quality of the coating was tested by measuring the equilibrium liquid height in each individual microcapillary for every batch of MCF strips coated.

Equilibrium liquid height.
For measuring the equilibrium liquid height in the coated MCF strips, the bottom end of a dried and clean glass capillary of MCF strip (at least 100 in length) was immersed in a transparent cuvette filled with Ultra-Pure water and the equilibrium position of the meniscus recorded as the vertical difference between the liquid level in the cuvette and the bottom of the meniscus in the capillary. This was repeated at least three times. Using the Laplace-Young equation, [2] the mean contact angle between the meniscus and the inner capillary surface were also calculated, being 671.3 degrees for the MCF (Table S2) and 50.9 degrees for the glass capillary.
The measured mean equilibrium capillary rise was 32.5mm for the glass capillary and 58±3.0mm for the PVOH coated MCF strips (Table S2 and Figure S2C).  For that, reagents were loaded into the MCF strips (for singleplex SARS-CoV-2-S1 immunoassay) using a push-fit play PEEK luer connector for MCF developed in-house 36 fitted to a plastic syringe, and for multiplex DENV immunoassays, antibodies (Table S1) injected into individual capillaries with 1mL syringe fitted with 29G needle (BD Microlance, UK).

Colorimetric and fluorescent breakthrough curves in automated MCF siphon. A hydrophilic
MCF strip (89mm in length) was fitted into the cassette shown in Figure 1C  board (IO Rodeo Inc., Pasadena, USA). Fluorescence signal for the strip was determined from the peak green channel pixel intensity using Image J (NIH, USA). Table S1. Serotype specific DENV NS1 antibody clone pairs (all these antibody reagents were manufactured in house) [3] Antibody clone Use Reactivity to DENV serotype Type of epitope Isotype Derivation of pressure balance model. Assuming steady-state operation and in the absence of an air-liquid interface inside the siphon (i.e. siphon fully-primed), fluid flow through a siphon microcapillary will experience two forces, being liquid pressure head (P H ) and pressure drop (P resistance ): As fluid flow is laminar, the term representing pressure drop through resistance can be replaced by the well-known Hagen-Poiseuille law: For a circular microcapillary with inner diameter d c , total length L, and configured as a 'beak swan' siphon with a net hydrostatic liquid head, H, the pressure balance can be re-written as: To yield flow rate Q discharged, evenly, through N parallel microcapillaries, Eq. (S3) can be rewritten as:

Ethical statement
This study was approved by the ethics committee of Siriraj Institutional Review Board, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand. The written formal consent was obtained from parents or guardians before enrolment of each patient.